Methods and compositions for impairing multiplication of HIV-1

ABSTRACT

A composition which elicits antibodies to multiple known variants of Tat protein of HIV-1 of both the B and non-B clades contains the peptide R1-Asp-Pro-Asn-Leu-Asp-Pro-Trp-Asn-R2 SEQ ID NO: 23, and preferably an additional at least two variants of a peptide or polypeptide of the formula: R1-Asp-Pro-Y 7 -Leu-Glu-Pro-Trp-Z 12 -R2 SEQ ID NO: 8. In this composition, at least one of the two variants contains Arg at Y 7  and Lys at Z 12 , and in at least a second of the two variants Y 7  is Asn and Z 12  is Asn. Vaccinal and pharmaceutical compositions can contain one or more such peptides associated with carrier proteins, associated in multiple antigenic peptides, or as part of recombinant proteins. Diagnostic compositions and uses are described for assessing the immune status of vaccinated patients.

FIELD OF THE INVENTION

The present invention relates generally to compositions and methodsuseful for inhibiting the multiplication of human immunodeficiencyvirus-1 (HIV-1) in infected patients, symptomatic or asymptomatic, andfor attenuating HIV-1 multiplication following primary infection inpreviously uninfected subjects, thus minimizing progression to AIDS.

BACKGROUND OF THE INVENTION

A variety of approaches to the treatment of human immunodeficiency virustype 1 (HIV-1) have focused on the transactivating (tαt) gene of HIV-1,which produces a protein (Tat) essential for transcription of the virus.The tαt gene and its protein have been sequenced and examined forinvolvement in proposed treatments of HIV [see, e.g., U.S. Pat. Nos.5,158,877; 5,238,882; and 5,110,802; International Patent ApplicationNos. WO92/07871, WO91/10453, WO91/09958, and WO87/02989, published May14, 1992, Jul. 25, 1991, Jul. 11, 1991 and May 21, 1987, respectively].Tat protein is released extracellularly, making it available to be takenup by other infected cells to enhance transcription of HIV-1 in thecells and by noninfected cells, to alter host cell gene activations. Tatrenders the cells susceptible to infection by the virus. Uptake of Tatby cells is very strong, and has been reported as mediated by a shortbasic sequence of the protein [S. Fawell et al., Proc. Natl. Acad. Sci.,USA, 91:664-668 (1994)].

Immunization with HIV-1 Tat protein as a potential AIDS vaccine is underactive investigation. The HXB/LAV HIV-1 Tat sequence has been used asthe immunogen in reported studies, either as a recombinant protein [A.Cafaro et al, Nat. Med. 5:643-650 (1999)], a DNA vaccine [S. Calarota etal, Lancet, 351:1320-5 (1998)], inactivated protein (Tat toxoid) [S. S.Cohen et al, Proc. Natl. Acad. Sci. USA, 96(19):10842-10847 (1999); A.Gringeri et al, J. Hum. Virol., 1:293-8 (1998)] or a DNA vaccineexpressing inactive Tat [E. Caselli et al, J. Immunol., 162:5631-5638(1999)]. Immunizations with the full Tat sequence induced both cellularand humoral immunity. See, also, M. C. Rhe et al, J. Acquir. ImmmuneDefic. Syndr. Hum. Virol. 10:408-416 (1995), C. J. Li et al, Proc. Natl.Acad, Sci. USA, 94:8116-8120 (1997); and others].

International Patent Application No. WO92/14755, published Sep. 3, 1992,relates to the Tat protein and to the integrin cell surface receptorcapable of binding to the Tat protein. Two Tat sequences that bindintegrin are identified: -Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg- [SEQ IDNO: 1], as well as -Gly-Arg-Gly-Asp-Ser-Pro- [SEQ ID NO: 2]. Thesesequences are the basic region or domain which is the dominant bindingsite for the integrin. This specification demonstrates that a number ofpeptides corresponding to these Tat sequences and the correspondingintegrins block in vitro cell binding to Tat coated plates, as doantibodies to the appropriate integrins. However, the specification alsoshows that these reagents do not block uptake of functional Tat by cells(see Example 9 in WO92/14755), thus nullifying the proposed mechanism ofaction for therapeutic benefit in HIV infection. The Tat sequencesdescribed in this international application are distinct from thepeptide immunogens of the present invention.

Both monoclonal and polyclonal antibodies to Tat protein have beenreadily produced in animals and shown to block uptake of Tat protein invitro [see, e.g., D. Brake et al, J. Virol., 64:962 (1990); D. Mann etal, EMBO J., 10: 1733 (1991); J. Abraham et al, cited above; P. Auron etal, cited above; M. Jaye et al, cited above; G. Zauli et al, citedabove]. More recent reports showed that monoclonal or polyclonalantibodies to Tat protein added to tissue culture medium attenuatedHIV-1 infection in vitro [L. Steinaa et al, Arch. Virol., 139:263(1994); M. Re et al, cited above; and G. Zauli et al, J. Acq. Imm. Def.Syndr. Hum. Retrovirol., 10:306 (1995)].

The inventor's own publications [G. Goldstein, Nature Med., 2:960(1996), and International Patent Application No. WO95/3 1999, publishedNov. 30, 1995] reviewed the evidence indicating that secretion of HIV-1Tat protein from infected cells and uptake by both infected anduninfected cells was important for the infectivity of HIV-1. Previousstudies also showed that antibodies to Tat protein in vitro blockeduptake of Tat and inhibited in vitro infectivity. Active immunization ofmammals was suggested to induce antibodies to HIV-1 Tat protein as apotential AIDS vaccine. See, also, G. Goldstein et al, “Minimization ofchronic plasma viremia in rhesus macaques immunized with synthetic HIV-1Tat peptides and infected with a chimeric simian/human immunodeficiencyvirus (SHIV₃₃)”, Vaccine, 18:2789 (2000).

Other publications by the inventor, International Patent Application No.WO99/02185, published Jan. 21, 1999, and U.S. Pat. No. 5,891,994, issuedApr. 6, 1999 (both incorporated by reference herein), revealed a newconcept in treatment and prevention of HIV-1 infection that utilized Tatsequences which were recognized as epitopes by the rabbit immune system.Unlike the prior disclosures discussed above, these publications relateto therapeutic and immunogenic combinations requiring at least two, andpreferably all four, of the Tat peptides or polypeptides comprising the“Epitope I” sequences spanning Tat amino acid residues 4 (or 5) through10, as follows: -Asp-Pro-X₇-Leu-Glu-Pro- [SEQ ID NO: 3] orR¹-Val-Asp-Pro-X₇-Leu-Glu-Pro-R² [SEQ ID NO: 4], wherein X₇ is Arg, Lys,Ser or Asn. Such compositions induce antibodies that react with mostHIV-1 Tat proteins and impair the multiplication of HIV-1. According tothis publication, certain other Tat sequences, which comprise an“Epitope II” peptide or polypeptide spanning Tat amino acid residues41-50 of the formula R3-Lys-X₄₂-Leu-Gly-Ile-Ser-Tyr-Gly-Arg-Lys-R4 [SEQID NO:5], wherein X₄₂ is selected from the group consisting of Gly orAla, may be added to this composition. Alternatively, an “Epitope III”peptide or polypeptide spanning Tat amino acid residues 56-62 of theformula R5-Arg-Arg-X₅₈-Z₅₉-A₆₀-Y₆₁-Ser-R6 [SEQ ID NO:6], wherein X₅₈ isselected from the group consisting of Ala, Pro Ser and Gln; wherein Y₆₁is selected from the group consisting of Asp, Asn, Gly and Ser; whereinZ₅₉ is selected from the group consisting of Pro and His; wherein A₆₀ isselected from the group consisting of Gln and Pro, may be added to thiscomposition. Still alternatively, an “Epitope IV” peptide or polypeptidespanning Tat AA residues 62-73 of the formulaR7-Ser-Gln-X₆₄-His-Gln-Y₆₇-Ser-Leu-Ser-Lys-Gln-Pro-R8 [SEQ ID NO:7],wherein X₆₄ is selected from the group consisting of Asn and Thr;wherein Y₆₇ is selected from the group consisting of Ala and Val, may beadded to this composition. The composition itself may be employed toinduce antibodies to a large number of Tat sequences characteristic ofthe multiple variants of HIV-1. The compositions or antibodies generatedare used as vaccine or prophylactic treatments against these multiplevariants.

Despite the growing knowledge about HIV-1 disease progression, thereremains a need in the art for the development of compositions andmethods for treatment of HIV-1, both prophylactically andtherapeutically, which are useful to lower the viral levels of HIV-1 forthe treatment and possible prevention of the subsequent, generallyfatal, AIDS disease.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides a composition comprisingat least two variants of a peptide or polypeptide of the Epitope Iformula R1-Asp-Pro-Y₇-Leu-X₉-Pro-Trp-Z₁₂-R2 [SEQ ID NO:8], wherein Y₇ isselected from the group consisting of Arg, Lys, Ser and Asn; wherein X₉is selected from the group consisting of Glu and Asp; wherein Z₁₂ isselected from the group consisting of Lys and Asn; wherein R1 isselected from the group consisting of hydrogen, a lower alkyl, a loweralkanoyl, and a sequence of between 1 to about 5 amino acids, optionallysubstituted with a lower alkyl or lower alkanoyl; wherein R2 is selectedfrom the group consisting of a free hydroxyl, an amide, and a sequenceof one or up to about 5 additional amino acids, optionally substitutedwith an amide. In this composition, at least one of the two variantsmust have the formula wherein Y₇ is Arg and Z₁₂ is Lys, and at least asecond of the two variants must have the formula in which Y₇ is Asn andZ₁₂ is Asn. Each peptide of this composition is recognized as an HIV-1Tat Epitope I by a primate immune system. This formula permits theconstruction and use of a variety of peptide combinations.

In another aspect, the above-described composition further contains oneor more additional peptide or polypeptide(s) which represent other aminoacid sequences which correspond to HIV-1 Tat amino acid residues 5through amino acid residue 12. These optional amino acid sequences aredescribed in detail below. These sequences are preferably from an HIV-1strain with a Tat protein variant at that location.

In another aspect, this invention provides a composition described abovethat contains peptides or polypeptides which comprise at least the tworequired Epitope I peptides, recognized by primates (and preferablyadditional Epitope I peptides), in combination with one or more HIV-1Tat Epitopes II, III and/or IV. Epitopes II, III and IV are the HIV-1Tat peptide formulae described in International Patent Publication No.WO99/02185, incorporated herein by reference. Such compositions cancombine appropriate HIV-1 Tat peptides, so as to provide for acomposition that induces antibodies reactive with greater than about 95%of all known HIV-1 Tat proteins.

In yet a further aspect, the invention provides an antibody compositioncomprising at least one antibody, preferably generated in a primate,which specifically binds to a peptide or polypeptide of the formulaR1-Asp-Pro-Y₇-Leu-X₉-Pro-Trp-Z₁₂-R2 [SEQ ID NO:8], wherein Y₇ isselected from the group consisting of Arg, Lys, Ser and Asn; wherein X₉is selected from the group consisting of Glu and Asp; wherein Z₁₂ isselected from the group consisting of Lys and Asn; wherein R1 isselected from the group consisting of hydrogen, a lower alkyl, a loweralkanoyl, and a sequence of between 1 to about 5 amino acids, optionallysubstituted with a lower alkyl or lower alkanoyl; wherein R2 is selectedfrom the group consisting of a free hydroxyl, an amide, and a sequenceof one or up to about 5 additional amino acids, optionally substitutedwith an amide. This antibody composition preferably comprises at leasttwo antibodies, i.e., one antibody which binds to the Epitope I variantin which Y₇ is Arg and Z₁₂ is Lys, and at least a second antibody whichbinds to a second Epitope I variant in which Y₇ is Asn and Z₁₂ is Asn.Other antibodies directed to other variants than the two specifiedvariants may also be included in this composition. These antibodies inthe composition bind to Epitope I sequences recognized by the primateimmune system, which epitope is present on multiple variants of HIV-1Tat proteins. These antibodies include a variety of antibody constructs,such as monoclonal antibodies, as described in detail below.

In still another aspect, the invention provides an antibody,particularly a monoclonal antibody, which specifically binds to aprimate-recognized epitope of an HIV Tat protein, the epitope comprisingthe amino acid sequence -Asp-Pro-Y₇-Leu-X₉-Pro-Trp-Z₁₂- [SEQ ID NO:9],wherein Y₇, X₉ and Z₁₂ are defined as above.

In yet another aspect, the invention provides an antibody compositioncomprising at least one antibody that recognizes Epitope II peptidesequence -Lys-X₄₂-Leu-Gly-Ile-Ser-Tyr-Gly-Arg-Lys- [SEQ ID NO: 10],where X₄₂ is Gly or Ala, as a distinct epitope from previously describedantibodies which recognize the epitope of-Leu-Gly-Ile-Ser-Tyr-Gly-Arg-Lys-[SEQ ID NO: 11]. Preferably, thecomposition comprises one antibody which recognizes both the peptide inwhich X₄₂ is Gly and the peptide in which X₄₂ is Ala. These antibodiesare preferably generated in primates. These antibodies in thecomposition bind to Epitope II sequences recognized by the primateimmune system, which epitope is present on multiple variants of HIV-1Tat proteins. These antibodies include a variety of antibody constructs,as described in detail below.

In still another aspect, the invention provides an antibody, preferablya monoclonal antibody, that recognizes Epitope II peptide sequence-Lys-X₄₂-Leu-Gly-Ile-Ser-Tyr-Gly-Arg-Lys- [SEQ ID NO: 10], where X₄₂ isGly or Ala, as a distinct epitope from the epitope of-Leu-Gly-Ile-Ser-Tyr-Gly-Arg-Lys- [SEQ ID NO: 11], recognized bypreviously described antibodies.

In yet a further aspect, the invention provides a recombinant orsynthetic gene which encodes sequentially a peptide or polypeptide thatcontains at least two variants of a peptide or polypeptide of theEpitope I formula R1-Asp-Pro-Y₇-Leu-X₉-Pro-Trp-Z₁₂-R2 [SEQ ID NO: 8], asdefined above. In this synthetic gene, at least one of the two variantsmust have the formula wherein Y₇ is Arg and Z₁₂ is Lys, and at least asecond of the two variants must have the formula in which Y₇ is Asn andZ₁₂ is Asn. Optionally, this synthetic gene comprises a carboxy terminalEpitope II peptide, as recognized by the primate immune system.Alternatively, the recombinant or synthetic gene contains the seven oreight preferred primate-recognized Epitope I amino acid sequencesidentified below. The synthetic gene may contain each amino acidsequence separated by a spacer sequence, or may express eachpeptide/polypeptide in an open reading frame with a carrier protein. Thesynthetic gene may be separated from the carrier protein by a spacer ifthe spacer is fused to a primate-recognized Epitope I sequence, leavingan Epitope II sequence at the carboxy terminus of the recombinantprotein. Further embodiments include multiple Epitope I peptides of theabove formula fused together and to the carrier protein.

In yet a further aspect, the invention provides a synthetic molecule,e.g., a vector, comprising the above-described synthetic gene,operatively linked to regulatory nucleic acid sequences which direct andcontrol expression of the product of the synthetic gene in a host cell.

In another aspect, the invention provides a recombinant microorganism,e.g., a virus or commensal bacterium, which contains the above describedsynthetic gene or synthetic molecule. This microorganism is capable ofexpressing multiple copies of the product of the gene or molecule in ahost.

Still another aspect of the invention is a pharmaceutical compositionuseful for inducing antibodies that react with a large number of knownHIV-1 Tat proteins, e.g., greater than 95%, and preferably greater than99%, of the known Tat proteins. These induced antibodies can impair themultiplication of HIV-1. The pharmaceutical composition comprises atleast one of the recombinant or synthetic peptide/polypeptidecompositions described above, or the synthetic gene/molecule describedabove; or the recombinant microorganism described above, in apharmaceutically acceptable carrier.

Still a further aspect of the invention is a pharmaceutical compositionuseful for impairing the multiplication of HIV-1, this compositioncontaining an above described antibody composition or monoclonalantibody composition.

In yet a further aspect of the invention, a method for reducing theviral levels of HIV-1 involves exposing a human or other primate toantibody-inducing pharmaceutical compositions described above, activelyinducing antibodies that react with most HIV-1 Tat proteins, andimpairing the multiplication of the virus in vivo. This method isappropriate for an HIV-1 infected subject with a competent immunesystem, or an uninfected or chronically infected, but asymptomatic,subject. The method induces antibodies which react with HIV-1 Tatproteins, and which reduce viral multiplication during any initial acuteinfection with HIV-1 and which further minimize chronic viremia whichleads to AIDS.

In still another aspect, the invention provides a method for reducingthe viral levels of HIV-1 by administering to a human, who is incapableof mounting an effective or rapid immune response to infection withHIV-1, a pharmaceutical composition containing the antibody compositionsdescribed above. The method can involve chronically administering thecomposition.

Yet other aspects of the invention include methods for producing thecompositions described above, as well as host cells transfected withsuch compositions.

Still another aspect of this invention is a kit useful for themeasurement and detection of titers and specificities of antibodiesinduced by immunization with the compositions described above. The kitof the invention includes preferably the two required Epitope I peptidesdescribed above, as well as addition peptides of the Epitope I,recognized by primates, and possibly additional peptides of Epitopes IIthrough IV, and coated solid supports, a labeled reagent for detectingthe binding of antibodies to these peptides, and miscellaneoussubstrates and apparatus for evoking or detecting the signals providedby the labels, as well as conventional apparatus for taking bloodsamples, appropriate vials and other diagnostic assay components.

In yet a further aspect, the invention provides a method for detectingthe titers and reactivity patterns of antibodies in subjects immunizedwith the compositions of this invention. The method includes the stepsof incubating dilutions of the subject's biological fluid, e.g. serum,with plates or beads on which are bound one or more peptides of theEpitope I sequences of this invention and optionally, the Epitopes IIthrough IV, washing away unbound biological materials, and measuring anyantibody binding to the peptides with labeled reagent, e.g., ananti-human immunoglobulin to which is associated an enzyme. Depending onthe type of label employed, the signal produced by the label may beevoked by further adding a substrate which reacts with the enzyme, e.g.,producing a color change. Other conventional labels may also beincorporated into this assay design.

Other aspects and advantages of the present invention are describedfurther in the following detailed description of the preferredembodiments thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a graph of ELISA titers of rabbit antiserum to larger linearEpitope I peptides on truncated detector peptides, expressed as apercentage of maximal binding to larger peptides. The N- or C-terminalamino acids of the corresponding detector peptides are shown below eachcolumn in single letter code.

FIG. 1B is a graph of ELISA titers of primate antiserum to larger linearEpitope I peptides on truncated detector peptides, expressed as apercentage of maximal binding to larger peptides. The N- or C-terminalamino acids of the corresponding detector peptides are shown below eachcolumn in single letter code.

FIG. 2A is a graph of ELISA titers of rabbit antiserum to linear EpitopeII peptides on truncated detector peptides, expressed as a percentage ofmaximal binding to larger peptides. The N- or C-terminal amino acids ofthe corresponding detector peptides are shown below each column insingle letter code.

FIG. 2B is a graph of ELISA titers of primate antiserum to larger linearEpitope II peptides on truncated detector peptides, expressed as apercentage of maximal binding to larger peptides. The N- or C-terminalamino acids of the corresponding detector peptides are shown below eachcolumn in single letter code.

FIG. 3A illustrates the design of a pentavalent Epitope I/Epitope IIHIV-1 Tat immunogenic construct in three letter amino acid code [SEQ IDNO: 12].

FIG. 3B illustrates the design of an octavalent universal Epitope I inthree letter amino acid code [SEQ ID NO: 13].

FIG. 3C illustrates the design of an univalent universal Epitope IIimmunogenic construct in three letter amino acid code [SEQ ID NO: 14].

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a solution to the above-stated problem byproviding additional compositions which induce antibodies in uninfectedor early stage HIV-1 infected subjects still capable of mounting animmune response to an immunogen, the antibodies reacting with a largenumber (i.e., greater than 95%, and preferably, greater than 99%) ofknown HIV-1 Tat protein variants. The term “Tat sequence (or protein)variant” means a polypeptide or peptide containing Tat protein aminoacid residues, or a sequence from another HIV-1 strain Tat protein thatis substantially similar to the consensus sequence of Table I [SEQ IDNO: 15]. Each variant may differ from the consensus sequence and/or fromanother variant by at least one amino acid change within the residues ofinterest for Epitopes I through IV. This change may provide the same ordifferent antigenic specificity to that particular Tat Epitope whenadded to the composition of the invention.

The antibodies induced by compositions of this invention can inhibitmultiplication of HIV-1, which prevents further disease progression toAIDS. Antibody compositions are also provided for use in infected ornon-infected humans, who are incapable of mounting an effective or rapidimmune response to HIV-1 infection. These compositions are capable ofreacting with large numbers of Tat proteins, thus reducing viral levelsof HIV-1. These antibodies are useful in both therapeutic andprophylactic contexts to control the development of AIDS in a largepopulation exposed to, or infected by, HIV-1 strains which produce uponinfection immunologically distinct Tat proteins.

The compositions of the present invention include peptides or proteins,based on peptides provided by an epitope of HIV-1 Tat protein to whichprimates developed antibodies, or nucleic acid sequences which encodethe peptides and polypeptides that induce antibodies to Tat in primates.These induced antibodies, in turn, impair multiplication of HIV-1.

HIV-1 Tat protein is produced from two exons: Exon 1 encodes a 72 aminoacid (AA) protein which may be expressed without splicing or which maybe spliced with the approximately 15-32 amino acids encoded by Exon 2.The HIV-1 Tat Exon I sequence appears in Table I, and is a consensussequence based on Tat protein sequences of 31 known HIV-1 strains foundin the common B subtype [NIH Los Alamos database]. The amino acidpositions in which variations appear are in lower case letters. In TableI [SEQ ID NO: 15], the amino acid residue at position 73 is the firstPro of Exon 2 of HIV-1 Tat. Since the 72 amino acid product of Exon 1 iscapable alone of cellular uptake and activation, it is essential thatantibodies react with and interdict intercellular transport of the 72amino acid peptide. HIV-1 Tat contains a cysteine-rich region between AApositions 22 and 37 of Exon 1 [SEQ ID NO: 15], with a single covalentbond between Cys₂₁ and Cys₃₇, producing a complex tertiary structure.The scientific literature has indicated that this region does not appearto be immunogenic. Predominant antibodies to Tat are to the linearN-terminal Pro-rich region (AA1-21) and to the linear basic region(AA44-65), with an additional antibody reported to AA62-73.

The inventor has previously identified epitopes, i.e., binding regions,recognized by rabbit antibodies (antigenic sequences) in the N-terminallinear sequence 1-21 (22 AA) of Exon 1 [SEQ ID NO: 15] of Tat variants,and had defined four B cell epitopes in HIV-1 Tat. As previouslydescribed in International Patent Publication WO99/02185, immunogenicregions of this larger sequence were recognized by the rabbit immunesystem. These regions are identified in the consensus sequence of Exon 1in Table I below: Epitope I was identified by rabbit antibodies as thenine amino acid sequence of AA positions 2-10 of Exon 1. Epitope II wasidentified as the eight amino acid sequence of AA positions 43-50 ofExon 1. Epitope III was identified as the 7 amino acid sequence of AApositions 56-62 of Exon 1. Epitope IV was identified as the twelve aminoacid sequence of AA positions 62-73 of Tat, including the first Pro (AA73) of Exon 2, and overlaps Ser 62 of Epitope III.

TABLE I Consensus Tat Sequence  1                                    10                                   20Met glu Pro Val asp pro arg Leu Glu Pro Trp lys His Pro Gly Ser Gln ProIys thr                                     30                                     40ala cys thr asn Cys Tyr Cys Lys lys Cys Cys phe his Cys gln val Cys Pheile thr                                       50                                   60Lys gly Leu gly Ile Ser Tyr Gly Arg Lys Lys Arg Arg Gln Arg arg arg alapro gln                                      70 asp Ser gln thr his Glnval ser Leu ser Lys gln [SEQ ID NO: 15]

In the present invention, however, the inventor has detected asurprising shift in the amino acid sequences of particularly Epitope Irecognized by B cells in primates. In Table I, Epitope I and IIsequences recognized by primates are underlined. Primate-recognizedEpitope I spans Tat amino acid residues 5 to 12. The sequence of EpitopeII recognized by B cells in primates spans amino acids 41-50. EpitopesIII and IV are the same epitopes recognized in rabbits, as reported inWO99/02 185, incorporated herein by reference.

A. Primate Recognized-Epitope I Immunogenic Compositions

In one embodiment, the present invention provides a compositioncontaining at least two variants of a peptide or polypeptide recognizedby the primate immune system, and eliciting a specific humoral immuneresponse (for the purpose of this invention) in a primate exposed to thesequences in vivo. These primate-recognized Epitope I amino acidsequences correspond to amino acid residues 5-12 of the Tat consensussequence [SEQ ID NO:15] of Table I, derived from a number of “Tatsequence variants”. Primate-recognized Epitope I defines peptides of thegeneral formula: R1-Asp-Pro-Y₇-Leu-X₉-Pro-Trp-Z₁₂-R2 [SEQ ID NO: 8],wherein Y₇ is Arg, Lys, Ser or Asn; X₉ is Glu or Asp; and Z₁₂ is Lys orAsn. This formula permits a variety of variant mammalian Epitope Ipeptides. The composition of this invention must contain at least onepeptide variant wherein Y₇ is Arg and Z₁₂ is Lys, and at least a secondpeptide variant in which Y₇ is Asn and Z₁₂ is Asn.

The specified amino acids appearing in the formula of primate-recognizedEpitope I above is a minimum reactive primate Epitope I sequence. Eachimmunogen defined by that formula which is employed in methods of thisinvention to raise antibodies to the minimum Epitope I sequence may be alarger amino acid sequence. For example, the minimum Epitope I aminoacids are flanked by other amino acids, so that the entire Epitope Iimmunogenic sequence is between 8 and about 25 amino acids in length.The identity of the flanking amino acids is not essential to thebiological function of the Epitope I immunogen. In particular,additional amino acids on the N-terminus of primate-recognized Epitope Isequences do not affect immunogenicity. Thus, for eachprimate-recognized Epitope I peptide, the N-terminal R1 may be a freehydrogen on the unmodified N terminal amino acid, or a lower alkyl(i.e., C1-C10 alkyl), or a lower C1-C10 alkanoyl, such as an acetylgroup. R1 may also include a sequence of between 1 to about 5 aminoacids, optionally substituted with a lower alkyl or lower alkanoyl.Preferably, R1 represents 2 amino acids. In one embodiment, RI is Val.In another embodiment, R1 is -X₂-Pro-Val-, wherein X₂ is Glu or Asp.Preferably, R1 represents 3 amino acids.

Additional amino acids on the C-terminus of the primate-recognizedEpitope I minimum sequence can enhance antibody titer. While theC-terminal R2 can be a simple free hydroxyl group on the C terminalamino acid, it can also be a C terminal amide. However, to enhancetiter, R2 is preferably a sequence of between 1 to about 14, preferablyabout 4 additional amino acids amidated at the carboxyl terminus. In apreferred embodiment, R2 is -His-Pro-Gly-Ser-amide [SEQ ID NO:16].

Preferably a composition of this invention includes in addition to thetwo required peptides identified above, at least five or six differentamino acid sequences of the primate-recognized Epitope I formula. Mostpreferably, the composition comprises seven or eight variant amino acidsequences, identified immediately below. The composition may alsocontain other peptide or polypeptide sequences, each containing adifferent X₉, Y₇ and Z₁₂ combination. As demonstrated in the examplesbelow, with three sites of antigenic variability in theprimate-recognized Epitope I, a preferred composition of this inventionmay contain sufficient peptides of primate-recognized Epitope I tocomprise 95% of all known B clade and non-B clade HIV-I Tat variants byincluding the two “required” peptides:

R1-Asp-Pro-Arg-Leu-Glu-Pro-Trp-Lys-R2 [SEQ ID NO:17]; and

R1-Asp-Pro-Asn-Leu-Glu-Pro-Trp-Asn-R2 [SEQ ID NO:18], as well as one tofive of the following additional Epitope I peptides:

R1-Asp-Pro-Lys-Leu-Glu-Pro-Trp-Lys-R2 [SEQ ID NO:19];

R1-Asp-Pro-Ser-Leu-Glu-Pro-Trp-Lys-R2 [SEQ ID NO:20];

R1-Asp-Pro-Asn-Leu-Glu-Pro-Trp-Lys-R2 [SEQ ID NO:21];

R1-Asp-Pro-Lys-Leu-Glu-Pro-Trp-Asn-R2 [SEQ ID NO:22]; and

R1-Asp-Pro-Asn-Leu-Asp-Pro-Trp-Asn-R2 [SEQ ID NO:23]; as well as stilloptionally, the rare variant R1-Asp-Pro-Ser-Leu-Glu-Pro-Trp-Asn-R2 [SEQID NO: 24].

The primate-recognized Epitope I composition of the invention maycontain a number of additional peptides or polypeptides that containother sequences which correspond to amino acid residues between AA 5 toAA 12 of SEQ ID NO:15, but are derived from other Tat variants which donot cross-react well with antibodies to the primate-recognized Epitope Icompositions. The Epitope I compositions of this invention may containmultiple copies of five or more different Epitope I peptides, in anyorder. In one embodiment, at least one copy of seven or all eight of theamino acid sequences described above [SEQ ID NOs: 17-24] are present.

These peptides or polypeptides of the invention are producedsynthetically or recombinantly. Optional amino acids (e.g., -Gly-Ser-)or other amino acid or chemical compound spacers may be included at thetermini of the peptides for the purpose of linking the peptides togetheror to a carrier. This composition may take the form of one or more ofthe above-described peptides expressed as a synthetic peptide coupled toa carrier protein. Alternatively, a composition may contain multipleEpitope I peptides, each expressed as a multiple antigenic peptide,optionally coupled to carrier protein. Alternatively, the selectedpeptides may be linked sequentially and expressed within a recombinantlyproduced protein. As one embodiment, the eight specifically identifiedsequences above are linked sequentially, with and without spacer aminoacids therebetween, to form a larger recombinant protein. Alternatively,the recombinant protein may be fused in frame with a carrier protein.These primate Epitope I compositions are designed to induce antibodiesreactive with greater than 95% of the known variants of the HIV-1 Tatprotein including Tat proteins of the HIV-1 B and non-B clades.

Primate-recognized Epitope I compositions demonstrate a biologicalactivity of inducing in an immunized, immune competent primate, i.e., anon-infected human, or an asymptomatic infected human, an active humoralimmune response (i.e., antibodies) that is directed against greater than95%, and preferably greater than 99%, of the known variants of Tatproteins of HIV-1. The end result of such treatment is an impairment ofthe multiplication of HIV-1 following an acute infection. Thisimpairment prevents high post-seroconversion plasma levels of HIV-1associated with progression to AIDS. Active induction of antibodies inthe asymptomatic phase of HIV infection may reduce viral multiplication,lower the plasma viral load and reduce the likelihood of progression toAIDS. The composition which contains at least the two requiredprimate-recognized Epitope I immunogens, and preferably seven or eightof those Epitope I sequences [e.g., SEQ ID NO: 17-24], can elicit animmune response to about 95% of 294 known Tat sequences of the common Bsubtypes of HIV-1 and with Tat proteins of all 56 non-B HIV-1 subtypesthat have been sequenced [courtesy of Dr. Esther Guzman, Los AlamosNIAID HIV database; GenBank database].

B. Immunogenic Compositions Containing Additional Epitopes

In another embodiment, the present invention provides other compositionswhich employ two or more primate-recognized Epitope I sequences combinedwith at least one Epitope II sequence and, optionally with one or moreEpitope III or IV peptides. These HIV-1 Tat Epitopes II, III and IV, asrecognized by the rabbit immune system, are described in detail inInternational Patent Application No. WO99/02185, incorporated herein byreference.

Briefly described, the Epitope II sequence elicits a specific humoralimmune response in a primate exposed to the Epitope II sequence in vivo.Epitope II, as recognized by primates, defines peptides of the formulaR3-Lys-X₄₂-Leu-Gly-Ile-Ser-Tyr-Gly-Arg-Lys-R4 [SEQ ID NO: 5], whereinX₄₂ is Gly or Ala. The minimum epitope recognized by the primate immunesystem is that of the specifically-identified amino acids of thatformula, i.e., -Lys-Gly-Leu-Gly-Ile-Ser-Tyr-Gly Arg-Lys- (amino acids41-50 of SEQ ID NO: 15). This is also the sequence of the presentlypreferred immunogen for Epitope II. This immunogen in which X₄₂ is Glyinduces antibodies cross-reactive with the sequence in which X₄₂ is Ala.This would react/cross-react with greater than 95% of known HIV-1 Tatproteins. This Epitope II sequence has no antigenic variability in alarge number of known HIV-1 Tat variants. The N terminal R3 mayrepresent the hydrogen on the unmodified N terminal amino acid Lys, orR3 may be a lower alkyl, or a lower alkanoyl, such as an acetyl group,substituent on the Lys. R3 may also include a sequence of between 1 toabout 5 amino acids, optionally substituted with a lower alkyl or loweralkanoyl. The C terminal R4 may represent the free hydroxyl of the Cterminal amino acid, or R4 may be an amide on that C terminal aminoacid. R4 may include additional non-polar amino acids, such as a spacerof sequence -Gly-Ser-Gly-Ser- [SEQ ID NO: 25] or some other amino acidsequence. However, R4 cannot be the basic amino acids -Lys-Arg-Arg-which naturally occur in the Tat sequence after the last amino acid inthe Epitope II formula. This Epitope II sequence found in 294 B cladeTat variants is recognized by primates (As reported in WO99/02185, therabbit immune system recognizes the epitope from AA43-50 of SEQ ID NO:15).

Epitope II is poorly immunogenic when presented within other sequences.Thus, for optimal immunogenicity, this sequence is prepared as asynthetic peptide fused to, or coupled to, a carrier protein or as amultiple antigenic peptide, optionally coupled to carrier protein.Alternatively, Epitope II may be expressed as the C terminal sequence ofa recombinant protein, which is optionally fused in frame to a carrierprotein at its amino terminal sequence. In a composition of thisinvention, an Epitope II peptide is preferably presented alone or incombination with one or more primate-recognized Epitope I peptides.

Briefly described and as identified in WO99/02185, Epitope III definespeptides of the formula: R5-Arg-Arg-X₅₈-Z₅₉-A₆₀-Y₆₁-Ser-R6 [SEQ ID NO:6], wherein X₅₈ may be Ala, Pro, Ser or Gln; Y₆₁ may be Asp, Asn, Gly orSer; Z₅₉ may be Pro or His; and A₆₀ may be Gln or Pro. Epitope IVdefines peptides of the formula:R7-Ser-Gln-X₆₄-His-Gln-Y₆₇-Ser-Leu-Ser-Lys-Gln-Pro-R8 [SEQ ID NO: 7],wherein X₆₄ may be Asn or Thr; and Y₆₇ may be Ala or Val.

Thus, the compositions of this invention, i.e., the peptide/polypeptidescontaining the above-identified amino acid sequences, when provided to ahuman subject, are useful in the immunologic interdiction ofextracellular Tat proteins of most HIV-1 strains. These compositionsfunction to critically reduce chronic multiplication of the virus andpermit effective immune control of the virus.

The immunogens for each Epitope are preferably designed to induceantibodies reactive with the highest proportion of naturally occurringvariants of each epitope. For an epitope such as primate-recognizedEpitope I, multiple copies of an immunogen could be incorporated in asynthetic or recombinant immunogen to enhance the immunogenicity andproduce higher titer antibodies. Furthermore, immunogens for two or moreepitopes could be combined to extend coverage, since variations insequence of each epitope occur independently. Thus, as one example, acomposition of this invention contains the two requiredprimate-recognized Epitope I peptides, as well as four or five of theother Epitope I peptides specifically identified above with a Cys on theterminus, which is coupled to carrier protein. Alternatively, multipleantigenic peptides may be prepared, optionally coupled to carrierprotein, and combined to form a composition of this invention.Alternatively, mixtures of two or more immunogens could be used.

The primate-recognized Epitope I immunogens of this invention, with orwithout any Epitope II, III or IV or other optional immunogens, may beprepared and used in immunogenic compositions in a variety of forms, forexample, chemically synthesized or as recombinant peptides,polypeptides, proteins, fusion proteins or fused peptides.

1. Recombinant or Synthetic Peptide/Proteins Coupled to a Carrier

As one embodiment, a composition of the present invention may be asynthetic or recombinantly-produced peptide, containing at least the tworequired primate-recognized Epitope I immunogenic amino acid sequences(as well as additional other Epitope I sequences) and also containingone or more Epitope II/III/IV immunogenic amino acid sequences, coupledto a selected carrier protein. In this embodiment of a composition ofthis invention, multiple above-described primate-recognized Epitope Iamino acid sequences with or without flanking sequences, may be combinedsequentially in a polypeptide and coupled to the same carrier.Alternatively, the Epitope I, II, III, or IV immunogens, may be coupledindividually as peptides to the same or a different carrier proteins,and the resulting immunogen-carrier constructs mixed together to form asingle composition. Such sequences may be made synthetically byconventional methods of chemical synthesis or recombinantly byexpression in a selected host cell, also by now-conventional means.

For this embodiment, the carrier protein is desirably a protein or othermolecule which can enhance the immunogenicity of the selected immunogen.Such a carrier may be a larger molecule which has an adjuvanting effect.Exemplary conventional protein carriers include, without limitation, E.coli DnaK protein, galactokinase (galK, which catalyzes the first stepof galactose metabolism in bacteria), ubiquitin, α-mating factor,β-galactosidase, and influenza NS-1protein. Toxoids (i.e., the sequencewhich encodes the naturally occurring toxin, with sufficientmodifications to eliminate its toxic activity) such as diphtheria toxoidand tetanus toxoid may also be employed as carriers. Similarly a varietyof bacterial heat shock proteins, e.g., mycobacterial hsp-70 may beused. Glutathione reductase (GST) is another useful carrier. One ofskill in the art can readily select an appropriate carrier.

In a particularly desirable immunogen-carrier protein construct, the tworequired Epitope I immunogens and three to six additionalprimate-recognized Epitope I immunogens and optional immunogenicpeptides/polypeptides may be covalently linked to a mycobacterial E.coli heat shock protein 70 (hsp70) [K. Suzue et al, J. Immunol., 156:873(1996)]. In another desirable embodiment, the composition is formed bycovalently linking the immunogen-containing peptide or polypeptidesequences to diphtheria toxoid.

2. Multiple Antigenic Peptide

In yet another embodiment, the peptides or polypeptide epitopeimmunogens and any selected optional immunogens may be in the form of amultiple antigenic peptide (“MAP”, also referred to as an octamericlysine core peptide) construct. Such a construct may be designedemploying the MAP system described by Tam, Proc. Natl. Acad. Sci. USA,85:5409-5413 (1988). This system makes use of a core matrix of lysineresidues onto which multiple copies of the same primate-recognizedEpitope I of the invention are synthesized as described [D. Posnett etal., J. Biol. Chem., 263(4):1719-1725 (1988); J. Tam, “ChemicallyDefined Synthetic Immunogens and Vaccines by the Multiple AntigenPeptide Approach”, Vaccine Research and Developments, Vol. 1, ed. W.Koff and H. Six, pp. 51-87 (Marcel Deblau, Inc., New York 1992)]. EachMAP contains multiple copies of only one peptide. Therefore, acomposition containing MAPs will contain at least two, and preferablyabout seven, MAPs. One MAP will have the first required peptide orpolypeptide Epitope I immunogen attached to each lysine core; a secondMAP will have the second required peptide or polypeptide Epitope Iimmunogen attached to each lysine core. Still other MAPs, each with adifferent primate-recognized Epitope I amino acid sequence identifiedabove, may be included. Multiple different MAPs may be employed toobtain any desired combination of Epitope II, III or IV sequences.Preferably these MAP constructs are associated with other T cellstimulatory sequences, or as pharmaceutical compositions, administeredin conjunction with T cell stimulatory agents, such as known adjuvants.

3. Spacers

In either of the above compositions, e.g., aspeptide/polypeptide-carrier constructs or MAPs, each peptide/polypeptideimmunogen, or each amino acid sequence in the immunogen, may beoptionally separated by an optional amino acid sequence called a“spacer”. Spacers are sequences of between 1 to about 4 amino acidswhich are interposed between two sequences to permit linkagetherebetween without adversely affecting the three dimensional structureof the immunogen. Spacers may also contain restriction endonucleasecleavage sites to enable separation of the sequences, where desired.Suitable spacers or linkers are known and may be readily designed andselected by one of skill in the art. Preferred spacers are sequencescontaining Gly and/or Ser amino acids.

F. Nucleic Acid Compositions of the Invention, Including a Synthetic orRecombinantly-Produced Gene

Other embodiments of this invention include nucleic acid sequences thatencode the above-described primate-recognized Epitope Ipeptide/polypeptide compositions, including the peptide and polypeptideimmunogens of the compositions described above, and including thosepeptides and polypeptides fused to carrier proteins. The nucleic acidsequences may also include sequences encoding the carrier proteins.

Thus, one preferred embodiment of the invention is a “synthetic gene”that encodes sequentially for at least the two requiredprimate-recognized Epitope I immunogenic peptides/polypeptides. Notethat while the gene is referred to as “synthetic”, it may be designed bychemical synthesis or recombinant means, as desired. The synthetic genepreferably encodes seven or all eight of the specifically identifiedprimate-recognized Epitope I amino acid sequences [SEQ ID NOS: 17-24].The synthetic gene can also encode any selection of an Epitope II or IIIimmunogen, provided that the Epitope II or III peptide is fused to the Cterminus of the primate-recognized Epitope I sequence and not furthermodified on its own C terminus. The synthetic gene may encode multiplecopies of the two required Epitope I amino acid sequences, or copies ofadditional multiple different immunogens or amino acid sequences, ormultiple copies of multiple different immunogens or amino acidsequences. The synthetic gene may encode the selected amino acidsequences in an open reading frame with, or fused to, a nucleic acidsequence encoding a carrier protein. A further characteristic of thesynthetic gene may be that it encodes a spacer between each sequenceencoding an immunogen and/or between the sequence encoding an immunogenand the sequence encoding the carrier protein.

The synthetic gene of the present invention may also be part of asynthetic or recombinant molecule. The synthetic molecule may be anucleic acid construct, such as a vector or plasmid which contains thesynthetic gene encoding the protein, peptide, polypeptide, fusionprotein or fusion peptide under the operative control of nucleic acidsequences encoding regulatory elements such as promoters, terminationsignals, and the like. Such synthetic molecules may be used to producethe polypeptide/peptide immunogen compositions recombinantly. Thesynthetic gene or synthetic molecule can be prepared by the use ofchemical synthetic methods or preferably, by recombinant techniques. Forexample, the synthetic gene or molecule may contain certain preferencecodons for the species of the indicated host cell.

The synthetic gene or molecule, preferably in the form of DNA, may beused in a variety of ways. For example, these synthetic nucleic acidsequences may be employed to express the peptide/polypeptides of theinvention in vitro in a host cell culture. The expressed immunogens,after suitable purification, may then be incorporated into apharmaceutical reagent or vaccine. Alternatively, the synthetic gene orsynthetic molecule of this invention may be administered directly into amammal, preferably a human, as so-called ‘naked DNA’ to express theprotein/peptide immunogen in vivo in a patient. See, e.g., J. Cohen,Science, 259:1691-1692 (Mar. 19, 1993); E. Fynan et al., Proc. Natl.Acad. Sci., USA, 90:11478-11482 (December 1993); and J. A. Wolff et al.,Biotechniques, 11:474-485 (1991), all incorporated by reference herein.The synthetic molecule, e.g., a vector or plasmid, may be used fordirect injection into the mammalian host. This results in expression ofthe protein by host cells and subsequent presentation to the immunesystem to induce antibody formation in vivo.

G. Microorganisms That Express The Synthetic Gene

In still another aspect of the present invention, the synthetic genes ormolecules of this invention may be incorporated into a non-pathogenicmicroorganism. The resulting microorganism, when administered to amammalian host, expresses and multiplies the expressed compositions ofthis invention in vivo to induce specific antibody formation. Forexample, non-pathogenic recombinant viruses or commensal bacterium whichcarry the compositions or synthetic genes of this invention and areuseful for administration to a mammalian patient may be prepared by useof conventional methodology and selected from among known non-pathogenicmicroorganisms.

Among commensal bacterium which may be useful for exogenous delivery ofthe synthetic molecule to the patient, and/or for carrying the syntheticgene into the patient in vivo, include, without limitation, variousstrains of Streptococcus, e.g., S. gordonii, or E. coli, Bacillus,Streptomyces, and Saccharomyces.

Suitable non-pathogenic viruses which may be engineered to carry thesynthetic gene into the cells of the host include poxviruses, such asvaccinia, adenovirus, adeno-associated viruses, canarypox viruses,retroviruses and the like. A number of such non-pathogenic viruses arecommonly used for human gene therapy, and as carriers for other vaccineagents, and are known and selectable by one of skill in the art.

H. Preparation or Manufacture of Compositions of the Invention

The compositions of the invention, and the individualpolypeptides/peptides containing the primate-recognized Epitope Iimmunogens of this invention and optionally one or more Epitope II, IIIor IV, the synthetic genes, and synthetic molecules of the invention,may be prepared conventionally by resort to known chemical synthesistechniques, such as described by Merrifield, J. Amer. Chem. Soc.,85:2149-2154 (1963). Alternatively, the compositions of this inventionmay be prepared by known recombinant DNA techniques by cloning andexpressing within a host microorganism or cell a DNA fragment carrying asequence encoding a peptide/polypeptide containing at least the tworequired primate-recognized Epitope I sequences with optional otherimmunogens and optional carrier proteins. Coding sequences for theEpitope I and optional immunogens can be prepared synthetically [W. P.C. Stemmer et al, Gene, 164:49 (1995)] or can be derived from viral RNAby known techniques, or from available cDNA-containing plasmids.

Combinations of these techniques may be used. For example, assembly ofsequential immunogens by conventional molecular biology techniques maybe used for production of the synthetic gene, and site-directedmutagenesis used to provide desired sequences of immunogens. The productof the synthetic gene is then produced recombinantly. All of thesemanipulations may be performed by conventional methodology.

Systems for cloning and expressing the peptide/polypeptide compositionsof this invention using the synthetic genes or molecules include the useof various microorganisms and cells which are well known in recombinanttechnology. These include, for example, various strains of E. coli,Bacillus, Streptomyces, and Saccharomyces, as well as mammalian, yeastand insect cells. Suitable vectors therefor are known and available fromprivate and public laboratories and depositories and from commercialvendors. Currently, the most preferred hosts are mammalian cells, suchas Chinese Hamster ovary cells (CHO) or COS-1 cells. These hosts may beused in connection with poxvirus vectors, such as vaccinia or swinepox.The selection of other suitable host cells and methods fortransformation, culture, amplification, screening and product productionand purification can be performed by one of skill in the art byreference to known techniques. See, e.g., Gething and Sambrook, Nature,293:620-625 (1981),. among others. Another preferred system includes thebaculovirus expression system and vectors.

When produced by conventional recombinant means, the compositions ofthis invention, i.e., the polypeptide/peptides containing the indicatedcopies of the primate-recognized Epitope I immunogens and optionalimmunogens may be isolated either from the cellular contents byconventional lysis techniques or from cell medium by conventionalmethods, such as chromatography. See, e.g., Sambrook et al., MolecularCloning. A Laboratory Manual., 2d Edit., Cold Spring Harbor Laboratory,New York (1989). Suitable plasmid and viral vectors used either forproduction of the peptide/polypeptide components as DNA vaccines arewell known to those of skill in the art and are not a limitation of thepresent invention. See, Sambrook et al., cited above and the referencesabove to production of the protein. See, also International PatentApplication PCT WO94/01139, published Jan. 20, 1994. Briefly, the DNAencoding the selected peptide/polypeptide is inserted into a vector orplasmid which contains other optional flanking sequences, a promoter, anmRNA leader sequence, an initiation site and other regulatory sequencescapable of directing the multiplication and expression of that sequencein vivo or in vitro. These vectors permit infection of the patient'scells and expression of the synthetic gene sequence in vivo orexpression of it as a protein/peptide or fusion protein/peptide invitro.

The resulting composition may be formulated into a primate-recognizedEpitope I composition with any number of optional immunogens andscreened for efficacy by in vivo assays. Such assays employ immunizationof an animal, e.g., a simian, with the composition, and evaluation oftiters of antibody to the Tat proteins of HIV-1 or to synthetic detectorpeptides corresponding to variant Tat sequences (as shown in theexamples below).

I. Antibody Compositions of the Invention

An antibody composition or ligand-binding composition of this inventionencompasses at least one antibody which specifically binds to an EpitopeI of the formula -Asp-Pro-Y₇-Leu-X₉-Pro-Trp-Z₁₂- [SEQ ID NO: 9], whereinY₇ is selected from the group consisting of Arg, Lys, Ser and Asn;wherein X₉ is selected from the group consisting of Glu and Asp; andwherein Z₁₂ is selected from the group consisting of Lys and Asn.Preferably, such an antibody composition includes at least two or moredifferent antibodies or ligands which specifically bind to at least thetwo required Epitope I sequences, as defined herein. Antibodies (orother binding ligands) are generated to the two required sequencesR1-Asp-Pro-Arg-Leu-Glu-Pro-Trp-Lys-R2 [SEQ ID NO: 17]; andR1-Asp-Pro-Asn-Leu-Glu-Pro-Trp-Asn-R2 [SEQ ID NO: 18] of Epitope I. Theantibodies of the composition thus bind an HIV Tat protein present onmultiple variants of HIV-1 Tat proteins. Additional antibodies orligands are generated to other sequences falling within the Epitope Iformula above.

In one embodiment, an isolated antibody directed which binds the EpitopeI peptide or polypeptide of the invention, as described above, is alsoan aspect of this invention. Such polyclonal antibody compositions aretypically produced by immunizing a mammal, preferably a primate, with apeptide/polypeptide composition containing the two required Epitope Iimmunogens, as well as an assortment of other primate-recognized EpitopeI immunogens and optional immunogens, as described above. Particularlydesirable as immunogens are a heptavalent primate-recognized Epitope Iimmunogen (i.e., without the rare variant) or an octavalent immunogen,such as the synthetic gene or fusion protein described in Example 3below, and/or a univalent Epitope II immunogen (i.e., a single EpitopeII peptide, optionally bound to a carrier). In additional to beinggenerated in primates, such antibodies may also be produced intransgenic animals, including so-called “humanized” transgenic mice.However, a desirable host for raising polyclonal antibodies to acomposition of this invention includes humans. The titer of suchpolyclonal antibodies raised in the mammal exposed to the Epitope Icompositions can be monitored by standard techniques, such as with anenzyme-linked immunosorbent assay. If desired, the antibody moleculescan be isolated from the mammal, e.g., from the whole blood, plasma orserum, and further purified from the plasma or serum of the immunizedmammal by conventional techniques. Conventional harvesting techniquescan include plasmapheresis, protein A chromatography, among others. Suchpolyclonal antibody compositions may themselves be employed aspharmaceutical compositions of this invention.

Alternatively, antibody producing cells may be obtained from the mammalsand used to prepare other forms of antibodies and ligands, e.g.,monoclonal antibodies, chimeric antibodies, humanized antibodies, humanantibodies, ligands produced by screening phage displays, antibodyfragments and mixtures thereof, and synthetic antibodies, monoclonalantibodies, chimeric antibodies, humanized antibodies and fully humanantibodies. Preparative techniques for generation of these types ofligands are known and the ligands themselves may be generated using thedisclosed amino acid sequences of the primate-recognized Epitope I andoptional immunogens. See, e.g., Kohler and Milstein (1975) Nature,256:495-497; Kozbor et al, (1983) Immunol. Today, 4:72; Cole et al,1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp.77-96; Harlow et al., Antibodies A Laboratory Manual, Cold Spring HarborLaboratory, (1988); Queen et al., Proc. Nat'l. Acad. Sci. USA,86:10029-10032 (1989); Hodgson et al., Bio/Technology. 9:421 (1991);International PCT Application PCT/GB91/01554, Publication No. WO92/04381and International PCT Application PCT/GB93/00725, Publication No.WO93/20210].

For example, in another embodiment, a monoclonal antibody specificallybinds to the minimum Epitope I sequence defined by-Asp-Pro-Y₇-Leu-X₉-Pro-Trp-Z₁₂- [SEQ ID NO: 9] of an HIV Tat protein:comprising any larger immunogen defined by the Epitope I formula, withthe variable amino acids and R groups as defined above. As oneembodiment, a monoclonal antibody binds specifically to the amino acidsequence -Asp-Pro-Asn-Leu-X₉-Pro-Trp-Asn- [SEQ ID NO:26], wherein X₉ isGlu or Asp. Still other monoclonal antibodies which bind specifically tothe minimum Epitope I sequences defined by the formula above are part ofthis invention.

In another embodiment of this invention, a monoclonal antibodyspecifically binds to a minimum Epitope II sequence comprising the aminoacid sequence -Lys-X₄₂-Leu-Gly-Ile-Ser-Tyr-Gly-Arg-Lys- [SEQ ID NO: 10],where X₄₂ is Gly or Ala, as a distinct epitope from the epitopeof-Leu-Gly-Ile-Ser-Tyr-Gly-Arg-Lys- [SEQ ID NO: 11], recognized bypreviously described antibodies. Preferably, an antibody compositioncomprises one antibody which is cross-reactive with both the peptide inwhich X₄₂ is Gly and the peptide in which X₄₂ is Ala. These antibodiesare preferably generated in primates. Still other monoclonal antibodieswhich bind specifically to the minimum Epitope II sequences defined bythe formula above are part of this invention.

Other anti-Tat antibodies may be developed by screening a recombinantcombinatorial immunoglobulin library (e.g., antibody phage displays)with primate-recognized HIV-1 Tat epitopes of this invention to isolateimmunoglobulin library members that bind to the HIV-1 Tat [W. D. Huse etal., Science, 246:1275-1281 (1988)]. Kits for generating and screeningphage display libraries are commercially available, e.g., PharmaciaRecombinant Phage Antibody System, Catalog No. 27-9400-01, StrategenePhage Display kits, etc. See, e.g., U.S. Pat. No., 5,223,409,International Publication No. WO92/09690, WO90/02809, etc. Chimericantibodies may similarly be developed using known techniques [Morrisonet al, (1984) Proc. Natl. Acad., Sci., USA, 81:6851; Takeda et al,Nature, 313:452(1984), among others]. Chimeric antibodies are moleculesin which different portions are derived from different animal species.Single chain antibodies may also be prepared by conventional methods[see, e.g., U.S. Pat. Nos. 4,946,778 and 4,704,692] using the variableportions of the polyclonal or monoclonal antibodies produced accordingto this invention. Antibody fragments, such as the Fab, F(ab′)₂ and Fvfragments and libraries thereof may also be employed in various aspectsof this invention.

These antibody/ligand compositions desirably bind to most known HIV-1Tat protein variants (e.g., greater than 95%, and preferably greaterthan 99% of known Tat protein variants), and prevent the Tat proteinsfrom supporting further HIV-1 multiplication. Such compositions caninclude a mixture of multiple different antibodies which bind HIV-1 Tatprotein epitope sequences from multiple strains of HIV-1. Thus, theseantibodies are useful in pharmaceutical methods and formulationsdescribed below.

J. Pharmaceutical Compositions of the Invention

As another aspect of this invention, a pharmaceutical composition usefulfor inducing antibodies that react with most (e.g., greater than 95%,preferably greater than 99%) known HIV-1 Tat proteins and impair themultiplication of HIV-1 can comprise as its active agents, at least thetwo required primate-recognized Epitope I peptides or polypeptides ofthis invention, and preferably additional Epitope I peptides. Severaldesirable compositions include the following above-described components:

(a) a peptide/polypeptide immunogen which contains at least the tworequired, and more preferably at least seven, of the primate-recognizedEpitope I amino acid sequences [SEQ ID NOS:17-24];

(b) a peptide/polypeptide immunogen of (a) which further contains any ofthe Epitope II, III or IV amino acid sequences, preferably a univalentEpitope II immunogen;

(c) a synthetic or recombinantly-produced gene encoding the two requiredprimate-recognized Epitope I sequences and preferably seven of theEpitope I sequences [SEQ ID NO: 17-24], and optional sequences asdescribed above;

(f) a synthetic molecule containing the synthetic gene of (c);

(g) a recombinant virus carrying the synthetic gene or moleculedescribed above; and

(h) a commensal bacterial carrying the synthetic gene or moleculedescribed above.

The selected active component(s) is present in a pharmaceuticallyacceptable carrier, and the composition may contain additionalingredients. Pharmaceutical formulations containing the compositions ofthis invention may contain other active agents, such as T cellstimulatory agents for the MAPs, adjuvants and immunostimulatorycytokines, such as IL-12, and other well-known cytokines, for theprotein/peptide compositions. All of these pharmaceutical compositionscan operate to lower the viral levels of a mammal.

As pharmaceutical compositions, the compositions comprisingprimate-recognized Epitope I peptide or nucleic acid sequences and theoptional immunogen sequences are admixed with a pharmaceuticallyacceptable vehicle suitable for administration to mammals forprophylaxis or treatment of virus infections. The proteins/peptides maybe combined in a single pharmaceutical preparation for administration.Suitable pharmaceutically acceptable carriers for use in an immunogenicproteinaceous composition of the invention are well known to those ofskill in the art. Such carriers include, for example, saline, bufferedsaline, a selected adjuvant, such as aqueous suspensions of aluminum andmagnesium hydroxides, liposomes, oil in water emulsions and others.Suitable adjuvants may also be employed in the protein-containingcompositions of this invention. Suitable vehicles for direct DNA,plasmid nucleic acid, or recombinant vector administration include,without limitation, saline, or sucrose, protamine, polybrene,polylysine, polycations, proteins, CaPO₄ or spermidine. See e.g, PCTapplication WO94/01139 and the references cited above. Thepeptide/polypeptide compositions and synthetic genes or molecules invivo are capable of eliciting in an immunized host mammal, e.g., ahuman, an immune response capable of interdicting multiple (e.g.,greater than about 95 to about 99%) known extracellular Tat proteinvariants from HIV-1 and thereby lowering the viral levels.

Yet another pharmaceutical composition useful for impairing themultiplication of HIV-1 comprises an antibody composition containing oneor more of the antibodies described in detail above. In a pharmaceuticalcomposition, the antibodies may be carried in a saline solution or othersuitable carrier. The antibody compositions are capable of providing animmediate, exogenously provided, interdiction of Tat.

The present invention is not limited by the selection of theconventional, physiologically acceptable, carriers, adjuvants, or otheringredients useful in pharmaceutical preparations of the types describedabove. The preparation of these pharmaceutically acceptablecompositions, from the above-described components, having appropriate pHisotonicity, stability and other conventional characteristics is withinthe skill of the art.

K. Method of the Invention—Impairing Multiplication of HIV-1

According to the present invention, a method for reducing the virallevels of HIV-1 involves exposing a human to the Tat antibody-inducingpharmaceutical compositions described above, actively inducingantibodies that react with multiple (e.g., greater than 95%, preferablygreater than 99% of the known) HIV-1 Tat proteins, and impairing themultiplication of the virus in vivo. This method is appropriate for anHIV-1 infected subject with a competent immune system, or for activeimmunization of an uninfected subject. The method induces antibodieswhich react with HIV-1 Tat proteins, reduce viral multiplication duringan initial acute infection with HIV-1 and minimize chronic viremialeading to AIDS. This method also lowers chronic viral multiplication ininfected subjects, again minimizing progression to AIDS. Use of thesemethods can control chronic HIV-1 infection, providing a novel mechanismof treatment not subject to the development of resistance. Theantibodies to Tat inhibit replication of HIV-1 quasispeciesindependently of the Tat that they are producing, since theextracellular Tat protein is not associated with the replicating moietyof the virus. Hence, there is no obvious mechanism by which Tatantibodies could generate selective pressure for non-reactive, escapeTat variants.

According to this method, the pharmaceutical compositions preferablycontain the peptide/polypeptide compositions, the synthetic genes ormolecules, the recombinant virus or the commensal recombinant bacterium.Preferably the compositions contain a heptavalent synthetic gene orfusion protein (without the rare variant of Epitope I) or the octavalentsynthetic gene or fusion protein of Example 3 and optionally a univalentEpitope II peptide. Each of these active components of thepharmaceutical composition actively induces in the exposed human theformation of anti-Tat antibodies which block the transfer of Tat frominfected cells to other infected or uninfected cells. This actionreduces the multiplicity of infection and blocks the burst of HIV-1viral expansion, and thus lowers viral levels. In already infectedpatients, this method of reduction of viral levels can reduce chronicviremia and progression to AIDS. In uninfected humans, thisadministration of the compositions of the invention can reduce acuteinfection and thus minimize chronic viremia leading to progression toAIDS.

Yet another aspect of the invention is a method for reducing the virallevels of HIV-1 by administering to a human incapable of mounting aneffective or rapid immune response to infection with HIV-1, apharmaceutical composition containing the antibody compositionsdescribed above. The method can involve chronically administering thecomposition. Among such patients suitable for treatment with this methodare HIV-1 infected patients who are immunocompromised by disease andunable to mount a strong immune response. In later stages of HIVinfection, the likelihood of generating effective titers of antibodiesis less, due to the immune impairment associated with the disease. Alsoamong such patients are HIV-1 infected pregnant women, neonates ofinfected mothers, and unimmunized patients with putative exposure (e.g.,a human who has been inadvertently “stuck” with a needle used by anHIV-1 infected human).

For such patients, the method of the invention preferably employs as thepharmaceutical composition an antibody composition of the invention. Theantibody composition includes a polyclonal antibody composition preparedin other mammals, preferably normal humans or alternatively, the otherforms of antibody described above, e.g., monoclonal, etc. These antibodycompositions are administered as passive immunotherapy to inhibit viralmultiplication and lower the viral load. The exogenous antibodies whichreact with multiple known Tat proteins from HIV-1 provide in the patientan immediate interdiction of the transfer of Tat from virally infectedcells to other infected or uninfected cells. According to this method,the patient may be chronically treated with the antibody composition fora long treatment regimen.

In each of the above-described methods, the compositions of the presentinvention are administered by an appropriate route, e.g., by thesubcutaneous, oral, intravenous, intraperitoneal, intramuscular, nasal,or inhalation routes. The presently preferred route of administration isintramuscular for the immunizing (active induction) compositions andintravenous (i.v.), subcutaneous (s.c.), or intramuscular (i.m.) for theantibody (passive therapy) compositions. A recombinant viral vector ornaked DNA is preferably administered intramuscularly; however, othercertain recombinant viral vectors and/or live commensal bacteria may bedelivered orally.

The amount of the protein, peptide or nucleic acid sequences of theinvention present in each vaccine dose is selected with regard toconsideration of the patient's age, weight, sex, general physicalcondition and the like. The amount of active component required toinduce an immune response, preferably a protective response, or producean exogenous effect in the patient without significant adverse sideeffects varies depending upon the pharmaceutical composition employedand the optional presence of an adjuvant (for the protein-containingcompositions).

Generally, for the compositions containing protein/peptide, fusionprotein, MAP or coupled protein, or antibody composition, each dose willcomprise between about 50 μg to about 20 mg of the peptide/polypeptideimmunogens per mL of a sterile solution. A more preferred dosage may beabout 500 μg of immunogen. Other dosage ranges may also be contemplatedby one of skill in the art. Initial doses may be optionally followed byrepeated boosts, where desirable.

The antibody compositions of the present invention can be employed inchronic treatments for subjects at risk of acute infection due to needlesticks or maternal infection. A dosage frequency for such “acute”infections may range from daily dosages to once or twice a week i.v.,s.c., or i.m., for a duration of about 6 weeks. The antibodycompositions of the present invention can also be employed in chronictreatments for infected patients, or patients with advanced HIV. Ininfected patients, the frequency of chronic administration may rangefrom daily dosages to once or twice per month i.v., s.c., or i.m., andmay depend upon the half-life of the immunogen (e.g., about 7-21 days).However, the duration of chronic treatment for such infected patients isanticipated to be an indefinite, but prolonged period.

Alternatively, compositions of this invention may be designed for directadministration of synthetic genes or molecules of this invention as“naked DNA”. As with the protein immunogenic compositions, the amountsof components in the DNA and vector compositions and the mode ofadministration, e.g., injection or intranasal, may be selected andadjusted by one of skill in the art. Generally, each dose will comprisebetween about 50 μg to about 1 mg of immunogen-encoding DNA per mL of asterile solution.

For recombinant viruses containing the synthetic genes or molecules, thedoses may range from about 20 to about 50 ml of saline solutioncontaining concentrations of from about 1×10⁷ to 1×10¹⁰ pfu/mlrecombinant virus of the present invention. A preferred human dosage isabout 20 ml saline solution at the above concentrations. However, it isunderstood that one of skill in the art may alter such dosages dependingupon the identity of the recombinant virus and the make-up of theimmunogen that it is delivering to the host.

The amounts of the commensal bacteria carrying the synthetic gene ormolecules to be delivered to the patient will generally range betweenabout 10³ to about 10¹² cells/kg. These dosages may be altered by one ofskill in the art depending upon the bacterium being used and theparticular composition containing Epitope I and optional immunogensbeing delivered by the live bacterium.

Thus, the compositions of this invention are designed to retard orminimize infection by the selected virus of an uninfected mammal, e.g.,human. Such compositions thus have utility as vaccines. Anti-Tat proteinantibodies are not reactive with the HIV-1 proteins used in diagnosticassays to detect seroconversion after infection. Thus, subjects treatedwith the compositions of this invention would not be stigmatized withfalse-positive tests for HIV-1 infection, and it would remain possibleto detect seroconversion if treated subjects did become infected withHIV-1.

Providing a mammal with the compositions of this invention, whether as aprotein/peptide-containing composition or by administration of a novelnucleic acid sequence encoding the immunogen, affords a radicallydifferent strategy for AIDS vaccination because it permits the loweringof viral levels by biological interdiction of desirably, greater thanabout 95%, and preferably greater than about 99%, of known Tat proteinvariants of HIV-1, lowering multiplication of HIV-1.

The use of the Tat immunogen-containing compositions has a particularlydesirable advantage in contrast to other treatments and prophylacticmethods employed against such viruses. Because interdiction of the Tatprotein extracellularly inhibits the multiplication of all HIVquasi-species or strains indiscriminately, it does not create aselective pressure on the parent virus itself for selection of mutantvirus variants. Thus, blocking the uptake of Tat protein by thepatient's cells not only reduces the level of viremia, but does so in amanner that precludes the selection of “escape variants”.

Additionally, the invention comprises a method of actively treatingasymptomatic HIV-1 infected subjects with viremia, since during thecourse of the disease, extracellular Tat protein likely contributes tothe persistent infection and immune abnormalities that are present atthis stage of HIV-1 infection. Interdiction of extracellular Tat proteinby antibodies induced by immunization according to this invention canreduce viremia with more effective immune control, and result in delayor prevention of progression to AIDS.

The mechanism of the present invention as described above is useful inimpeding the course of viral infection and producing desirable clinicalresults. More specifically, the compositions of this invention arecapable of reducing viremia in patients already infected with the virusby blocking further uptake of the Tat protein by uninfected cells. Thecompositions of the present invention, used either alone or inconjunction with other therapeutic regimens for HIV infected patients,are anticipated to assist in the reduction of viremia and prevention ofclinical deterioration.

For such therapeutic uses, the formulations and modes of administrationare substantially identical to those described specifically above andmay be administered concurrently or simultaneously with otherconventional therapeutics for the specific viral infection. Fortherapeutic use or prophylactic use, repeated dosages of the immunizingcompositions may be desirable, such as a yearly booster or a booster atother intervals.

L. Diagnostic Kits of this Invention

The peptides and polypeptides described above can also be employed asreagents of a kit useful for the measurement and detection of titers andspecificities of antibodies induced by vaccination with the compositionsdescribed above. The kit of the invention can include at least the tworequired Epitope I peptides identified above, and preferably two or moreof the primate-recognized Epitope I and optional immunogens. In oneembodiment, each peptide has on its N terminus the protein biotin and aspacer, e.g.,-Ser-Gly-Ser-Gly- [SEQ ID NO: 27]. Alternatively, thepeptide may have on its C terminus a spacer, e.g., -Gly-Ser-Gly-Ser-[SEQ ID NO: 25], and the protein biocytin. These embodiments enable thepeptides to be bound to an avidin-coated solid support, e.g., a plate orbeads. Other binding agents known to those of skill in the diagnosticassay art may also be employed for the same purposes. Also provided inthe kit are labeled reagents which detect the binding of antibody to theimmobilized Epitope peptides, such as a goat anti-human immunoglobulinor the like. The label on the reagent may be selected from the manyknown diagnostic labels, such as radioactive compounds, fluorescentcompounds and proteins, colorimetric enzymes, etc. The kit thus alsocontains miscellaneous reagents and apparatus for reading labels, e.g.,certain substrates that interact with an enzymatic label to produce acolor signal, etc., apparatus for taking blood samples, as well asappropriate vials and other diagnostic assay components. One of skill inthe art may also readily select other conventional diagnostic componentsfor this kit.

Such kits and reagents may be employed in a method for detecting thetiters and reactivity patterns of antibodies in subjects vaccinated withthe compositions of this invention. A method for determining thepresence and or titer of antibodies induced by immunization to a Tatimmunogen includes the steps of contacting a biological sample from animmunized subject, e.g., a body fluid, preferably blood, serum orplasma, but also possibly urine, saliva and other fluids or tissue, withone or more of the binding sequences of primate-recognized Epitope I andoptional immunogens, preferably immobilized on a solid support, such asa plate or beads. The primate-recognized Epitope I and optional bindingsequences employed in this method may be the unmodified minimal epitopebinding regions.

Once the biological sample is exposed to the immobilized peptides for asufficient time, the support is washed to eliminate any material fromthe biological sample which is not bound to the peptides. Such washingsteps are conventional in diagnostic assays, and performed with saline.If antibodies to Epitopes I and optional immunogens or a combinationthereof, were induced in the subject by the above-described treatment,the immobilized peptides have been bound with antibody from thebiological sample. Thereafter, a labeled reagent is added to thematerial on the support to detect the binding between the peptides onthe solid support and antibody in said biological sample. Preferably,such a reagent is an anti-human immunoglobulin, such as goat anti-humanimmunoglobulin. The label is selected from among a wide array ofconventionally employed diagnostic labels, as discussed above. In oneembodiment, the label can be a calorimetric enzyme, which upon contactwith a substrate produces a detectable color signal. The presence and/orintensity of the color provides evidence of the induction of antibody inthe treated subject. This assay may be employed to determine theefficacy of the immunization, as well as to monitor immune status of apatient.

The selection of particular assay steps, as well as a variety ofdetectable label systems, is well within the skill of the art. Suchselection is routine and does not limit the present invention.

M. Advantages of the Invention

One of the advantages of the compositions of this invention is the smallnumber of immunogens required for inclusion into a composition of thisinvention to cross-react with greater than 95 to greater than 99% ofknown Tat protein variants of HIV-1 of the common B subtype. Asillustrated in the examples below, the primate-recognized Epitope Iimmunogenic composition containing the two required primate-recognizedEpitope I amino acid sequences as well as the six additional Epitope Isequences cross-reacts with 95% Tat proteins of HIV-1 of the common Bsubtype, as well as with all 56 Tat protein sequences from less frequentnon-B subtypes of HIV-1. Thus, a single composition may be usefullyemployed in protecting against or treating infection, caused by the vastmajority of HIV-1 strains that can be encountered.

Further, having identified the precise epitopes of Tat against whichbinding is desired (i.e., AA5-12 of SEQ ID NO: 15) new desirable Tatpeptide immunogens from newly occurring HIV-1 strains or newlydiscovered strains may be easily identified using the methods describedherein, and included in the compositions. This flexibility enables thecompositions of this invention to be useful prophylactically against anynew strain or strains of HIV-1 identified in the future. In view of theteachings herein, one of skill in the art is expected to be readily ableto incorporate new combinations of Tat immunogens (and the nucleic acidconstructs encoding them) into the compositions.

For example, the use of conventional techniques such as PCR and highdensity oligonucleotide arrays [M. J. Kozal et al, Nature Med., 2:753(1996)] enables one of skill in the art to obtain the amino acidsequences of a large array of HIV-1 Tat proteins representing variantsof clinical isolates of HIV-1 strains and subtypes. Using suchtechniques permits determination of other variants of the ^(HIV-)1 Bsubtype as well as other subtypes in underdeveloped countries, whichhave not been so intensively studied to date. The determination of newTat sequences will enable ready inclusion of the corresponding peptidesas immunogens into compositions of this invention, allowing theinduction of an antibody response against other rare Tat proteins ofHIV-1.

Cross-reactivity studies with antibodies raised to synthetic peptidescorresponding to each Tat variant can be utilized to eliminate the needfor immunizing with Tat variants in which the sequence changes areimmunologically silent, in that these peptides are strongly bound byantibodies to the consensus sequence or other variants.

The following examples illustrate preferred methods for preparing thecompositions of the invention and utilizing these compositions to induceantibodies to Tat proteins of the virus in an immunized host. Theseexamples are illustrative only and do not limit the scope of theinvention.

EXAMPLE 1—IMMUNOLOGICAL STUDIES ON MINIMAL TAT PROTEIN AMINO ACIDSEQUENCES NECESSARY FOR BINDING TO ANTIBODY FOR PRIMATE-RECOGNIZEDEPITOPE I IN HIV-1 TAT PROTEIN, SEQUENCE VARIATIONS, AND IMMUNOLOGICALCROSS-REACTIVITIES OF ANTISERUMS TO THESE SEQUENCES

A. Synthetic Peptide and Conjugates

The synthetic peptides were synthesized by solid phase synthesis onderivatized polyethylene supports [R. M. Valerio et al, Int. J. PeptideRes., 44:158-165 (1994)]. Immunizing peptides were synthesized with anamino terminal Cys being incorporated to facilitate coupling to acarrier protein and an amidated C-terminus. Detector peptides weresynthesized with an amino terminal biotin-Ser-Gly-Ser-Gly- sequence [SEQID NO: 27] and a free acid function at the C-terminus for use in ELISAassays for detection of reactivity and cross-reactivity. Immunizingpeptides, covalently conjugated to diphtheria toxoid (DT) carrierprotein via the cysteinyl side chain, with a peptide-carrier ratio of5-8 [A. C. J. Lee et al., Molec. Immunol, 17:749 (1980)], were purifiedby high pressure liquid chromatography (HPLC) to greater than 95% purityby analytical HPLC and mass spectrometry, with detector peptides beingused at greater than 50% purity.

B. Immunizations

The peptide conjugates were taken up in purified water and emulsified1:1 with complete Freund's adjuvant (CFA) or incomplete Freund'sadjuvant (IFA) [ANTIBODIES—A LABORATORY MANUAL, Eds. E. Harlow and P.Lane, Cold Spring Harbor Laboratory (1998)]. Total volume per primatewas 1 ml, and this contained 100 μg of peptide coupled to DT.

Rhesus monkeys that were part of a viral challenge study were immunizedwith antigen in IFA/CFA as follows. Several primates were used for theimmunizing peptide, with the initial intramuscular (IM) injection withconjugate in CFA and a subsequent IM boost at 2 weeks with conjugate inIFA. A pre-bleed was drawn before the first injection and larger bleedswere taken 3 and 5 weeks after the booster injection.

C. ELISA Titers

ELISA assays were performed as described by H. M. Geysen et al., Proc.Natl. Acad. Sci. USA, 81:3998 (1983). Antibody titer was the reciprocalof the serum dilution that resulted in an absorbence 1.0 OD units abovebackground. The geometric mean titer (GMT) for 2-3 serums was calculatedfor each response, or single serums only were available for some monkeyimmunizations.

ELISA results for this assay in rabbits vs. monkeys are shown in FIGS.1A and 1B, respectively. The ELISA results demonstrated that the primateantibodies to the immunogen incorporating Epitope I were reacting withthe sequence -Asp-Pro-Arg₇-Leu-Glu₉-Pro-Trp-Lys₁₂- [AA5-12 of SEQ ID NO:15]. As discussed below, the positions 7, 9 and 12 represent commonvariants of this Epitope I peptide recognized by primates.

D. Analysis of Amino Acid Sequence Diversity Within the Epitopes

HIV-1 Tat first exon sequences were retrieved from GenBank and the LosAlamos Human Retroviruses and AIDS databases [HUMAN RETROVIRUSES andAIDS 1996, published by the Theoretical Biology and Biophysics Group ofthe Los Alamos National Laboratory, Los Alamos, NM, and additionalsequences kindly obtained from GenBank by Esther Guzman of the LosAlamos Laboratory]. Incomplete sequences and sequences with stop codonsor base deletions leading to a frameshift were deleted, as wereobviously identical repeat sequences from the same isolation. Variationsof amino acids at the positions within the epitopes were recorded andtabulated.

E. Antigenic Cross-Reactivities Between Variants

Antisera to the epitope consensus sequence were titered by ELISA on theconsensus sequence and on sequences with common amino acid variants todetermine the effects of amino acid polymorphisms on antigenicity.

F. Variations in Sequences

The primate-recognized Epitope I consensus sequences were evaluated formaximal frequency and recognition by primate antibodies. The antigenicand sequence conservation in HIV-I Tat proteins from 294 HIV-1 Tatproteins from 294 B lade (I) viruses and 56 non-B lade (II) viruses wereevaluated for the epitopes and the results tabulated in Tables IIthrough VI below.

The top row of Tables II and III indicates the consensus sequence formaximal frequency. The middle rows contain the percent incidence ofamino acids found in greater than 5% of sequences at each position, ifmultiple. The bottom row of each table is the total incidence includingamino acids occurring in greater than 5% of sequences, if multiple. Allof these selections in Table II create antigenically distinct epitopes(<25% cross-reactivity); and all of the selections in Table III, exceptfor the entries under amino acid 4.

TABLE II Epitope I - 294 B clades Val₄ Asp₅ Pro₆ Arg₇ Leu₈ Glu₉ Pro₁₀Trp₁₁ Lys₁₂ Arg (73) Lys (96) Lys (12) Asn (2) Ser (11) Asn (4) 100% 98%100% 100% 100% 100% 100% 100% 100%

TABLE III Epitope I - 56 Non B clades Val₄ Asp₅ Pro₆ Asn₇ Leu₈ Glu₉Pro₁₀ Trp₁₁ Asn₁₂ Val Asn (79) Glu Asn (87) (89) (86) Ile Lys (14) AspLys (13) (11) (14) Ser(5) 100% 100% 100% 98% 96% 100% 98% 100% 100%

As shown in Tables II and III, primate-recognized Epitope I has apotential 16-fold antigenic polymorphism, but one major antigen existsfor the B clades and another major antigen exists for the non-B clades.Five other variants account for greater than 95% of known Tat variants.See Tables IV and V; sequences indicated with an asterisk arerepresented in both B and non-B clades. Table IV—B Clades (294sequences).

TABLE IV B Clades (294 sequences) Percent Primate epitope sequence SEQID NOs Incidence Incidence ValAspProArgLeuGluProTrpLys AA4-12 of SEQ 22075 ID NO: 15 ValAspProLysLeuGluProTrpLys* AA185-193 of 35 12 SEQ ID NO:12 ValAspProSerLeuGluProTrpLys AA120-127 of 20 7 SEQ ID NO: 12ValAspProAsnLeuGluProTrpLys* AA55-63 of 7 2 SEQ ID NO: 12 Total: 282Total: 96 ValAspProArgLeuGluProTrpAsn 28 1 <1

TABLE V Non-B Clades (56 sequences) Percent Primate epitope sequence SEQID NOs Incidence Incidence ValAspProAsnLeuGluProTrpAsn AA227-235 of 3664 SEQ ID NO: 13 ValAspProLysLeuGluProTrpAsn AA344-352 of 8 14 SEQ IDNO: 13 ValAspProAsriLeuAspProTrpAsn 29 6 11 ValAspProAsnLeuGluProTrpLys*AA55-63 of 3 5 SEQ ID NO:13 ValAspProLysLeuGluProTrpLys* AA185-193 of 12 SEQ ID NO:12 Total: 53 Total: 95 ValAspProSerLeuGluProTrpAsn AA279-287of 1 2 SEQ ID NO: 13 ValAspProSerLeuAspProTrpAsn 30 1 2ValAspProAsnLeuAspProTrpLys 31 1 2

Table VI shows the poor antigenic cross-reactivity of the position 7variants of Epitope I, the antigenic distinction of the position 9variants and the extreme lack of cross-reactivity of antisera toGluProValAspProAsn₇LeuGlu₉ProTrpAsn₁₂ [AA225-235 of SEQ ID NO: 13] withGluProValAspProArg₇LeuGlu₉ProTrpLys₁₂ [AA 2-12 of SEQ ID NO: 15]containing variants at both positions 7 and 12. It further showsantigenic distinction of Glu9 and Asp9 variants.

TABLE VI ELISA Reactivity of monkey antiserums to Epitope I Immunogenson Detector peptides with Tat amino acid positions 7, 9 and 12Variations Immunogen epitope Detector peptide epitope sequence sequenceTiter (% of titer of self peptide) GluProVal GluProVal GluProValGluProVal GluProVal AspProAsn ₇ AspProAsn ₇ AspProArg ₇ AspProLys ₇AspProSer ₇ LeuGlu₉Pro LeuGlu₉Pro LeuGlu₉Pro LeuGlu₉Pro LeuGlu₉ProTrpLys₁₂ TrpLys12 TrpLys₁₂ TrpLys₁₂ TrpLys₁₂ [AA53-63 of [AA53-63 of[AA2-12 of [AA183-193 [AA105-115 SEQ ID NO: SEQ ID NO: SEQ ID NO: of SEQID of SEQ ID 12] 12] 15] NO: 12] NO: 12] 119,000 (100) 25,000 (21)24,000 (20) 24,000 (20) GluProVal GluProVal GluProVal GluProValAspProAsn ₇ AspProAsn ₇ AspProAsn ₇ AspProArg ₇ LeuGlu₉Pro LeuGlu₉ProLeuGlu₉Pro LeuGlu₉Pro TrpAsn ₁₂ TrpAsn ₁₂ TrpLys₁₂ TrpLys₁₂ [AA225-235[AA225-235 [AA53-63 of [AA2-12 of of SEQ ID of SEQ ID SEQ ID NO: SEQ IDNO: NO: 13] NO: 13] 12] 15] 157,000(100) 23,000(15) 2000(1) GluProValGluProVal GluProVal AspProAsn AspProAsn ₇ AspProAsn ₇ LeuGlu ₉Pro LeuGlu₉Pro LeuAsp ₉Pro TrpLys₁₂ TrpLys₁₂ TrpLys₁₂ [AA53-63 of [AA53-63 of [SEQID NO: SEQ ID NO: SEQ ID NO: 32] 12] 12] 163,000 (100) 6000 (4)

EXAMPLE 2—IMMUNOLOGICAL STUDIES ON MINIMAL TAT PROTEIN AMINO ACIDSEQUENCES NECESSARY FOR BINDING TO ANTIBODY FOR PRIMATE-RECOGNIZEDEPITOPE II IN HIV-1 TAT PROTEIN, SEQUENCE VARIATIONS, AND IMMUNOLOGICALCROSS-REACTIVITIES OF ANTISERUMS TO THESE SEQUENCES

Using the same procedures outlined in Example 1, the incidence of aminoacid sequence variation for 294 B clade and 56 non-B clade HIV-1 Tatsequences was determined within the Epitope II boundaries of antibodybinding in monkeys. The results are reported in Tables VII and VIII. Thetop lines of the tables contain the consensus sequence. The middle linescontain the percent incidence of amino acids found in greater than 5% ofsequences at each position, if multiple. The bottom line shows the totalincidence including amino acids occurring in greater than 5% ofsequences, if multiple. The amino acid variants at Tat position 42 wereantigenically cross-reactive.

TABLE VII Epitope II- 294 B clades Lys₄₁ Gly₄₂ Leu₄₃ Gly₄₄ Ile₄₅ Ser₄₆Tyr₄₇ Gly₄₈ Arg₄₉ Lys₅₀ Gly (72) Ala (28) 100% 100% 99% 99% 100% 98% 99%100% 99% 100%

TABLE VIII Epitope II- 56 Non-B clades Lys₄₁ Gly₄₂ Leu₄₃ Gly₄₄ Ile₄₅Ser₄₆ Tyr₄₇ Gly₄₈ Arg₄₉ Lys₅₀ 100% 100% 100% 99% 100% 95% 100% 100% 98%100%

As revealed in Tables VII and VIII, Epitope II shows almost completeantigenic conservation.

ELISA reactivity of monkey antiserums to Epitope II immunogen ondetector peptides with Tat Gly₄₂ or Ala₄₂ (variant) within the detectorsequences were measured and reported in Table IX below. See FIGS. 2A and2B for a graphical comparison of results in rabbits vs. monkeys,respectively.

TABLE IX Immunogen epitope Detector peptide epitope sequence sequenceTiter (% of titer on self peptide) LysGlyLeuGlyIle LysGlyLeuGlyIleSerTyrLysAlaLeuGlyIleSerTyr SerTyrGlyArgLys GlyArgLys GlyArgLys [AA41-50 ofSEQ [AA41-50 of SEQ [SEQ ID NO:33] ID NO: 15] ID NO: 15] 25,000 (100%)19,000 (76%)

EXAMPLE 3—DEVELOPMENT OF HUMAN MONO CLONAL ANTIBODY TREATMENT FORASYMPTOMATIC HIV-1 INFECTIONS

Commercially available antibody humanized mice are immunized with asuitable amount of the Epitope II immunogen:Cys-Gly-Ser-Lys-Gly-Leu-Gly-Ile-Ser-Tyr-Gly-Arg-Lys-amide [SEQ ID NO:34]coupled to diphtheria toxoid carrier protein. Hybridomas are screened onbiotin- Ser-Gly-Ser-Gly-Leu-Gly-Ile-Ser-Tyr-Gly-Arg-Lys-OH [SEQ ID NO:35] on streptavidin-coated plates, and an IgG monoclonal antibody withsubnanomolar binding affinity and no binding to complement receptors isselected. Specificity is confirmed on recombinant HIV-1 Tat protein.

Since the monoclonal antibody is directed to a non-self antigen, aconventional pre-clinical production, purification and safety testing isanticipated. Human monoclonal antibodies have a half-life of 20 days inman vs. the 18 hours half-life of OKT3, a mouse monoclonal antibodywhich is extensively consumed on internal CD3. Daily doses of 5 mg OKT3maintain trough levels around 1 microgram/ml in man. Thus, a biweeklydose of 5 mg anti-Tat monoclonal antibody is anticipated to besufficient to maintain similar trough levels, a greater than fifty-foldmolar excess over the estimated maximal circulating levels of up to 1ng/ml for HIV-1 Tat protein in infected subjects.

Control of plasma viral loads is now an accepted criterion of efficacyfor HIV-1 treatment. The efficacy of an anti-Tat monoclonal antibody canbe rapidly determined in asymptomatic HIV-1 infected subjects, initiallywith a four week course of treatment. This protocol is useful inuntreated patients, in patients that have failed in HAART protocols forvarious reasons, or in patients controlled by HAART therapy, withwithdrawal of this therapy for 4 weeks (viral loads rebound rapidly ifHAART is stopped). A 2 to 3 log reduction in plasma viral loads to belowthe LOD (50 viral RNA copies/mL) supports monotherapy with themonoclonal antibody, which is evaluated over a longer time-span.Reduction over one log (90%), but not below the LOD suggests the use ofthe monoclonal antibody as a component in therapy.

EXAMPLE 4—DEVELOPMENT OF A UNIVERSAL VACCINE TO PREVENT PROGRESSION TOAIDS IN SUBJECTS

A. Construction of a Synthetic Gene

FIG. 3A illustrates a synthetic gene encoding four copies each of thefour polymorphs of Epitope I detected for the rabbit antibody response,plus four copies of Epitope II, expressed in E. coli as a linear fusionprotein with E. coli DnaK (HSP70). This expressed protein contained allthe antigenic epitopes when tested in ELISA with epitope specific rabbitantiserums. However, when used to immunized in rabbit or monkey, allEpitope I variants were immunogenic, but Epitope II was not. Thus,Epitope II is best used as a synthetic peptide conjugate coupled to anappropriate carrier protein.

FIG. 3B illustrates a novel octavalent synthetic gene constructed toincorporate in frame eight primate-recognized Epitope I polymorphs,based on the polymorphism within the Epitope I boundaries recognized inprimates:

R1-Asp-Pro-Arg-Leu-Glu-Pro-Trp-Lys-R2 [SEQ ID NO: 17]

R1-Asp-Pro-Lys-Leu-Glu-Pro-Trp-Lys-R2 [SEQ ID NO: 19]

R1-Asp-Pro-Lys-Leu-Glu-Pro-Trp-Asn-R2 [SEQ ID NO: 22]

R1-Asp-Pro-Ser-Leu-Glu-Pro-Trp-Lys-R2 [SEQ ID NO: 20]

R1-Asp-Pro-Ser-Leu-Glu-Pro-Trp-Asn-R2 [SEQ ID NO: 24]

R1-Asp-Pro-Asn-Leu-Glu-Pro-Trp-Lys-R2 [SEQ ID NO: 21]

R1-Asp-Pro-Asn-Leu-Glu-Pro-Trp-Asn-R2 [SEQ ID NO: 18] and

R1-Asp-Pro-Asn-Leu-Asp-Pro-Trp-Asn-R2 [SEQ ID NO: 23]

The Epitope I sequences are separated by dipeptide spacers containingGly and/or Ser residues. The gene is assembled as described in W. P. C.Stemmer et al., Gene, 164:49 (1995). Briefly, top strand 60-meroligonucleotides (oligos) and bottom strand oligos with 20 nucleotide(nt) overlaps are synthesized along with two end 50-mers. The 60-mersare incubated together under hybridizing conditions and polymerase chainreaction (PCR) is used to fill in the sequence and amplify it. The end50-mers are then added and the assembly completed by PCR, with isolationof the full length gene on agarose gel. The gene is sequenced and foundto have the correct sequence within the actual epitopes. A similarheptavalent gene may be constructed by eliminating the rare variantR1-Asp-Pro-Ser-Leu-Glu-Pro-Trp-Asn-R2 [SEQ ID NO: 24].

B. Expression of the Fusion Protein

This gene described above is then excised with restriction enzymes andinserted into a suitable expression vector containing, in frame, thesequence for diphtheria toxoid (HSP70). E. coli are transfected andcolonies expressing the protein are isolated. The isolated colonies aregrown and expression is induced. Protein from colonies expressing thefusion protein are identified. The resulting protein is purifiedutilizing conventional methods.

FIG. 3C illustrates a monovalent Epitope II immunogen optionallyprepared as a conjugate of synthetic peptide with a carrier protein suchas diphtheria toxoid, using similar techniques.

C. Assays for Assessing the Expression of the Epitopes Correctly in theFusion Protein and Efficacy in Inducing Anti-Tat Antibodies

Four variants of Epitopes I, in which Y₇ is either Arg, Asn, Lys or Ser,and X₉ is Glu and Z₁₂ is Lys, and both variants of Epitope II areconstructed in a synthetic gene and expressed as a fusion protein, asdescribed in paragraphs A and B above. To determine if each epitope isexpressed in the fusion protein in a form that can be recognized byprimate antisera, primate antiserums generated to synthetic peptidescorresponding to the Epitope I sequences are tested by ELISA, usingconventional methodology. Plates are initially directly coated with thefusion protein and then exposed to 100 μg/ml solution of antiserums(e.g., rabbit antisera) which are known to be reactive for Epitopes Iand II. The variant epitope sequences are expressed in a conformationrecognizable by antibodies to the corresponding synthetic peptides, asshown by a titer of greater than 32,000 for each epitope.

To evaluate the immunogenicity of the multivalent immunogen, monkeyswere immunized with the fusion protein in IFA. The monkey antisera wasthen assessed on synthetic peptides of the Epitopes I and II.Significant titers developed to the Epitope I variants, i.e., titer of28,000 to Epitope I, when Y₇ is Arg and Z₁₂ is Lys; titer of 16,000 toEpitope I when Y₇ is Asn and Z₁₂ is Lys; titer of 37,000 to Epitope I,when Y₇ is Lys and Z₁₂ is Lys; and titer of 4,000 to Epitope I, when Y₇is Ser and Z₁₂ is Lys. Titer to Epitope II was 700, indicating that thisepitope is best presented for immunization as a synthetic peptidecoupled to a carrier.

EXAMPLE 5: METHOD AND KITS FOR DETECTING TITERS AND SPECIFICITIES OFANTIBODIES INDUCED BY VACCINATION

To follow the titer and specificities of antibodies induced followingimmunization with the vaccines of this invention, an assay method may beemployed. In one embodiment of such as assay, peptides containing theprimate-recognized Epitope I sequences reported in Example 1 (dependingon the composition of the immunizing vaccine) are used to develop kitsmeasuring titers and reactivity patterns of antibodies in vaccinatedsubjects.

These peptides are synthesized with Biotin-Ser-Gly-Ser-Gly- [SEQ IDNO:36] at the N-terminus. Each peptide is coated onto separate avidincoated plates, with a sequence -Ser-Gly-Ser-Gly- [SEQ ID NO:27] servingas a spacer to ensure that the relevant peptide sequence is external tothe biotin binding pocket of avidin. The plates are then incubated withdilutions of test serum, washed, and the antibody binding determinedwith reagent to human immunoglobulin, e.g., goat anti-humanimmunoglobulin, directly labeled with enzyme. A reagent is employed toreact with the enzyme and produce a calorimetric signal (R&D kitinserts).

Numerous modifications and variations of the present invention areincluded in the above-identified specification and are expected to beobvious to one of skill in the art. Such modifications and alterationsto the compositions and processes of the present invention are believedto be encompassed in the scope of the claims appended hereto.

What is claimed is:
 1. A composition comprising a peptide or polypeptideof the formula R1-Asp-Pro-Asn-Leu-Asp-Pro-Trp-Asn-R2 SEQ ID NO: 8,wherein R1 is selected from the group consisting of hydrogen, a loweralkyl, a lower alkanoyl, and a sequence of between 1 to about 5 aminoacids, optionally substituted with a lower alkyl or lower alkanoyl; andwherein R2 is selected from the group consisting of a free hydroxyl, anamide, and a sequence of one or up to about 5 additional amino acids,optionally substituted with an amide.
 2. The composition according toclaim 1 wherein R1 is Val, resulting in the sequenceVal-Asp-Pro-Asn-Leu-Asp-Pro-Trp-Asn of SEQ ID NO:
 8. 3. The compositionaccording to claim 1 wherein R1 is X₂-Pro-Val, wherein X₂ is selectedfrom the group consisting of Glu and Asp, optionally substituted with alower alkyl or lower alkanoyl, resulting in the sequenceX₂-Pro-Val-Asp-Pro-Asn-Leu-Asp-Pro-Trp-Asn SEQ ID NO:
 38. 4. Thecomposition according to claim 1 wherein R2 is -His-Pro-Gly-Ser-,resulting in the sequenceVal-Asp-Pro-Asn-Leu-Asp-Pro-Trp-Asn-His-Pro-Gly-Ser SEQ ID NO:16,wherein said carboxy-terminal Ser is optionally substituted with anamide.
 5. The composition according to claim 1 wherein said peptides orpolypeptides are produced synthetically.
 6. The composition according toclaim 1 wherein said peptides or polypeptides are producedrecombinantly.
 7. The composition according to claim 1 wherein one ormore of said peptides is expressed as a synthetic peptide coupled to acarrier protein.
 8. The composition according to claim 1 wherein one ormore of said peptides is expressed as a multiple antigenic peptide,optionally coupled to a carrier protein.
 9. The composition according toclaim 1 wherein one or more of the selected peptides is expressed withina recombinantly produced protein, optionally fused in frame with acarrier protein.
 10. The composition according to any of claims 7-9,wherein said carrier protein is selected from the group consisting of anE. coli DnaK protein, a GST protein, a mycobacterial heat shock protein70, a diphtheria toxoid, a tetanus toxoid, a galactokinase, anubiquitin, an α-mating factor, a β-galactosidase, and an influenza NS-1protein.
 11. A pharmaceutical composition comprising a composition ofclaim 1, a pharmaceutically acceptable carrier and an optional adjuvant.12. The composition according to claim 1, further comprising at leasttwo variants of a peptide or polypeptide of the formula selected fromthe group consisting of: R1-Asp-Pro-Y₇-Leu-Glu-Pro-Trp-Z₁₂-R2 SEQ ID NO:8, wherein Y₇ is selected from the group consisting of Arg, Lys, Ser andAsn; wherein Z₁₂ is selected from the group consisting of Lys and Asn;wherein R1 is selected from the group consisting of hydrogen, a loweralky, a lower alkanoyl, and a sequence of between 1 to about 5 aminoacids, optionally substituted with a lower alkyl or lower alkanoyl; andwherein R2 is selected from the group consisting of a free hydroxyl, anamide, and a sequence of one or up to about 5 additional amino acids,optionally substituted with an amide, and wherein at least one saidvariant is R1-Asp-Pro-Arg-Leu-Glu-Pro-Trp-Lys-R2 SEQ ID NO: 17, and theother said variant is R1-Asp-Pro-Asn-Leu-Glu-Pro-Trp-Asn-R2 SEQ ID NO:18.
 13. The composition according to claim 12, further comprising apeptide in which R1 is Val.
 14. The composition according to claim 12,further comprising a peptide in which R1 is X₂-Pro-Val, wherein X₂ isselected from the group consisting of Glu and Asp, optionallysubstituted with a lower alkyl or lower alkanoyl.
 15. The compositionaccording to claim 12 further comprising a peptide wherein R2 is-His-Pro-Gly-Ser-, wherein said carboxy-terminal Ser is optionallysubstituted with an amide.
 16. The composition according to claim 12comprising one or more additional sequences selected from the groupconsisting of: R1-Asp-Pro-Lys-Leu-Glu-Pro-Trp-Lys-R2 SEQ ID NO: 19R1-Asp-Pro-Lys-Leu-Glu-Pro-Trp-Asn-R2 SEQ ID NO: 22R1-Asp-Pro-Ser-Leu-Glu-Pro-Trp-Lys-R2 SEQ ID NO: 20R1-Asp-Pro-Ser-Leu-Glu-Pro-Trp-Asn-R2 SEQ ID NO: 24 andR1-Asp-Pro-Asn-Leu-Glu-Pro-Trp-Lys-R2 SEQ ID NO: 21, wherein R1 isselected from the group consisting of hydrogen, a lower alkyl, a loweralkanoyl, and a sequence of between 1 to about 5 amino acids, optionallysubstituted with a lower alkyl or lower alkanoyl; and wherein R2 isselected from the group consisting of a free hydroxyl, an amide, and asequence of one or up to about 5 additional amino acids, optionallysubstituted with an amide.
 17. The composition according to claim 16,further comprising at least seven of said amino acid sequences.